Current paradigms propose that in the mammalian Golgi, PI4P acts primarily as a precursor to PI4,5P2 (PIP2), which is an important plasma membrane regulator. We found that PI4P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase (PI4K), to determine if PI4P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI4P, blocks the recruitment of clathrin adaptor AP-1 complexes to the TGN and also blocks AP-1 independent export of constitutively secreted proteins from the TGN. The AP-1 recruitment defect is rescued by adding back PI4P but not PIP2. In addition, purified AP-1 binds PI4P, and anti-PI4P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that (i) PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI4P-rich domains that specify Arf recruitment of the AP-1 coat machinery; (ii) PI4KIIalpha regulates constitutive secretion by supporting PIP2 synthesis, because the VSVG export defect in PI4KIIalpha RNAi cells can be rescued by adding back either PI4P or PIP2. Aim I. Determine if AP-1 recruitment to the Golgi is mediated through AP-1 binding to PI4P. Potential PI4P binding residues in the AP-1 I mu subunit will be mutated, and the functional consequences of these mutations will be examined in vivo and in vitro. In vivo assays include determining if the mutant subunit has diminished ability to rescue the Golgi phenotype of mu 1 -/- fibroblasts. Aim II. Examine the role of PI4P in regulating the Golgi recruitment of other coat proteins and use time-lapse fluorescence imaging to characterize the effects of changing PI4P levels on the dynamic behavior of TGN-derived transport carriers. Aim III. Determine if PI4KIIlbeta, an Arfrecruited Golgi PI4K, overlaps functionally with PI4KIIalpha in AP-1 recruitment and VSVG export. PI4KIIlbeta will be overexpressed to determine if it rescues the PI4KIIalpha RNAi Golgi phenotypes, and PI4KIIlbeta will be knocked down by RNAi to determine if it inhibits secretion at the same or a different step as PI4KIIalpha RNAi. We will determine if phosphatidylinositol 4 phosphate 5 kinase beta (PI5PKbeta) regulates VSVG export downstream of these Golgi PI4Ks.